The present invention relates to methods and kits for specific binding assays, e.g., immunoassays, involving binding pair members on suspendable particles.
Immunoassays involving suspendable particles (e.g., polystyrene latex) are well known, with the presence of analyte binding pair member affecting the binding of particles to one another. In manual assays, the agglutinated particles are observed or separate by gravity. In U.S. Pat. No. 4,202,665 to Wenz, et al (1980) and in copending application 733,688 of Jensen, et al., agglutinated particles act specifically in a centrifugal field. In copending application 733,689 of Lentrichia, et al, particles of two specific gravities bind one to the other in an amount affected by the analyte concentration. When first particles of one specific gravity are moved by centrifugal action, those second particles of the other specific gravity which are bound move with the first particles. See also U.S. Pat. No. 4,115,535 to Giaver (1978) for assays having two particles, each having a protein binding to the same analyte.
U.S. Patent 4,459,361 to Gefter (1984) discloses a particle immunoassay in which particles bind or agglutinate with other particles as a function of analyte ligand concentration. Filtration (e.g., through a 0.4 micrometer pore size filter) allows single particles (e.g., of 0.3 micrometer diameter) to pass through while duplexes and higher aggregates of particles are trapped by the filter. Particles passing through the filter can be visualized, or visibility can be enhanced by color, optical density, fluorescence or other properties of the particles or by enzymes attached to the particles (col. 4, line 42 - col. 5, line 4).
U.S. Pat. No. 4,424,279 to Bohn, et al (1984) discloses an assay method and plunger apparatus wherein large particles (20-30 micrometers diameter) bearing a binding pair member (e.g., antibody) are located in an apparatus compartment. Analyte binding pair member (e.g., antigen) and soluble labeled binding pair member (e.g., second antibody-enzyme) each enter the apparatus compartment for a reaction period and then liquids are withdrawn from the apparatus compartment by vacuum, with the particles retained by a coarse (e.g., 15-17 micrometer pore size) filter. Thereafter, particles remaining in the apparatus compartment are assayed for labels e.g., by addition of enzymatic reagents and cleaving the linkage between the enzyme and the second antibody.
Several references including L.A. Blankstein, et al., Am. Clin, Prod. Rev. 33-41 (Nov. 1985) and U.S. Pat. Nos. 4,200,690 to Root, et al. (1980), 4,246,339 to Cole, et al (1981) and 4,407,943 (1983) describe immunoassays in which an antibody or antigen is immobilized in a microporous membrane have pore size significantly large (0.65-5.0 micrometer) for either small molecules or small molecules and bacteria to migrate through the membrane. Thus, in U.S. Pat. No. 4,200,690, an antibody coated on such a membrane captures analyte antigen from crude sample. After washing, a second antibody conjugated to an enzyme (soluble conjugate) is introduced to bind specifically to bound analyte antigen. After washing, enzymatic read-out is compared to that from a similar membrane with an antibody thereon not specific for the analyte. U.S. Pat. No 4,407,943 to Cole, et al (1983) is similar.
U.S. Pat. No. 4,486,530 to David, et al, discloses immunoassays with monoclonal antibodies, especially of a sandwich geometry. Reference is made, however, at col. 15, lines 33-51 to an assay employing chromophore labeled antigen and monoclonal antibody bound to latex so that analyte antigen inhibits agglomeration and thus permits the chromophore to remain in solution where its fluorescence will not be quenched. Other inhibition assays where one binding pair member may be particle-bound are described in U.S. Pat. No. 4,308,026 to Mochida (1981).